| Apple ring rot is a main disease in apple producing areas in China,which is a fungal disease.It mainly does harm to the stems and fruits,and it is a major threat to fruit production.Therefore,it is very important to study the relationship between apple fruit and apple ring rot.Through studying and analyzing the mechanism of the apple disease resistance and immunity,we analyzed the physiological activities and stress responses of apple infected by the pathogen,and excavated apple disease resistance gene.Meanwhile,we mastered the mechanism of the apple disease resistance.Apple disease resistance gene is the basis on the varieties improvement,and it is of vital significance of disease resistant varieties breading and apple quality and efficient production.At the same time,the study played a critical role in deep understanding of apple's disease resistance gene.Besides,it provided a scientific and technological support for the cultivation of the new disease resistant germplasm resources in the apple,and improved the development of apple industries in China.In the study,we chosen ‘Fuji' and ‘Golden Delicious',which were different textures in apple fruits,as research materials.After being inoculated with the pathogen,we studied the resistance by measuring ethylene release,hardness,gene relative expression in the apple,and further studied on ethylene biosynthesis related genes,cell wall softening genes and PR family genes.The results showed that the related genes were highly expressed by being inoculated with the pathogen,and it is a significant difference between the two different cultivars at the aspects of the resistance to the disease in the apple.Additionally,the resistance response to apple was complex in different developmental stages.For the expression of disease duration related genes in apple disease resistance,we selected the MdPR5 and the MdPR8 from apple fruits,and got the cDNA sequence of gene in apple through the extraction of plant RNA,PCR technology,genes cloning and other methods,then we based our analysis on the sequence,gene expression and gene transfer.Lastly,we verified the expression of ethylene genes responded to various apples by methods of molecular biology.The main results were as follows:1.Plant active oxygen burst under the stress.We chosen ‘Gala' tissue culture seedling as materials,which were treated by apple ring rot and flg22,detected the burst of reactive oxygen species by DAB staining method.So,we found that there were the obvious yellow particles on leave treated by apple ring rot and flg22,and the more yellow particles,the more active oxygen burst.We also found that the leave and the plants occurred on the adverse reactions when the plants' stem were flushed after flg22 treatment.2.When ‘Fuji' and ‘Golden Delicious' were inoculated with the pathogen,ethylene release rate,hardness change and lesion size were both have the obvious difference at the young fruit stage,the growth stage,and the mature stage.Meanwhile,the effect of inhibiting ethylene production in the fruits treated by 1-MCP was different at different stages in different species,and there was the most significant change in the mature period in Golden Delicious.After inoculation,Golden Delicious fruit spots and hardness were significantly less than Fuji.When we did qRT-PCR analysis for genes related to ethylene biosynthesis,such as MdERF2,MdACS4,MdCTR1,Md ACS5 a,MdACS5b,the results showed that the expressions,the MdACS5 a and the Md ACS5 b,increased thousands of times in the induction of the pathogen at the mature stage.Besides,1-MCP showed the significantly inhibited on the expression of the MdACS5 a and the MdACS5 b,and 1-MCP on the role of MdACS4 was significant in the mature period of sixth days in the Golden Delicious.3.We studied the qRT-PCR analysis for cell wall softening gene,like AFS1,PG1,MdPR5,MdPR8 and MdPR4,at different stages in ‘Fuji' and ‘Golden Delicious' inoculated with the pathogen.We found that all genes showed the high expressions and this proved the PRs possessed the disease resistance.4.We cloned two disease related genes named the MdPR5 and the MdPR8,then we analyzed the expression,the sequences,the original nuclear induction and the SDS-PAGE.All could devote to the basic work for the apple disease resistance gene and the improvement of apple varieties.Forecast,we found the amino acid sequence of MdPR5 in Arabidopsis,apple,pear,and quince displayed high similarity of some sequence by comparative analysis of amino acid sequences. |
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